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Screening for Microtubule-Disrupting Antifungal Agents by Using a Mitotic-Arrest Mutant of Aspergillus nidulans and Novel Action of Phenylalanine Derivatives Accompanying Tubulin Loss. Tetsuo Kiso, 2004.The microtubule, which is one of the major targets of anthelmintics, anticancer drugs, and fungicides, is composed mainly of  - and ß-tubulins . We focused on a unique characteristic of an Aspergillus nidulans benA33 mutant to screen for microtubule-disrupting antifungal agents . This mutant, which has a ß-tubulin with a mutation of a single amino acid, undergoes mitotic arrest due to the formation of hyperstable microtubules at 37°C . The heat sensitivity of the mutant is remedied by some antimicrotubule agents . We found that an agar plate assay with the mutant was able to distinguish three types of microtubule inhibitors . The growth recovery zones of the mutant were formed around paper disks containing microtubule inhibitors, including four benzimidazoles, ansamitocin P-3, griseofulvin, and rhizoxin, on the agar plate at 37°C . Nocodazole, thiabendazole, and griseofulvin reversed the mitotic arrest of the mutant and promoted its hyphal growth . Ansamitocin P-3 and rhizoxin showed growth recovery zones around the growth-inhibitory zones . Benomyl and carbendazim also reversed mitotic arrest but produced weaker growth recovery than the aforementioned drugs . Other microtubule inhibitors, such as colchicine, Colcemid, paclitaxel, podophyllotoxin, TN-16, vinblastine, and vincristine, as well as some cytoskeletal inhibitors tested, did not show such activity . In our screening, we newly identified two mycotoxins, citrinin and patulin, two sesquiterpene dialdehydes, polygodial and warburganal, and four phenylalanine derivatives, arphamenine A, L-2,5-dihydrophenylalanine (DHPA), N-tosyl-L-phenylalanine chloromethylketone, and N-carbobenzoxy-L-phenylalanine chloromethyl ketone . In a wild-type strain of A . nidulans, DHPA caused selective losses of microtubules, as determined by fluorescence microscopy, and of both
- and ß-tubulins, as determined by Western blot analysis . This screening method involving the benA33 mutant of A . nidulans is useful, convenient, and highly selective . The phenylalanine derivatives tested are of a novel type of microtubule-disrupting antifungal agents, producing an accompanying loss of tubulins, and are different from well-known tubulin inhibitors affecting the assembly of tubulin dimers into microtubules .
Multiple Nonidentical Reductive-Dehalogenase-Homologous Genes Are Common in Dehalococcoides. Tina Hölscher, 2004.Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH) genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene- and dioxin-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2 . The amplicons (1,350 to 1,495 bp) corresponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments (approximately 90 bp) of genes encoding putative membrane-anchoring proteins . Cloning and restriction analysis revealed the presence of at least 14 different RDH genes in each strain . All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases . Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range . All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp . strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively . The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195 . Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes . In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains . Unique Degradation Signal for ClpCP in Bacillus subtilis. Qi Pan, 2003.Regulation of the cell-specific transcription factor  F in the spore-forming bacterium Bacillus subtilis involves the antisigma factor SpoIIAB . Contributing to the activation of
F is the degradation of SpoIIAB in a manner that depends on the protease ClpCP . Here we show that the three residues (LCN) located at the extreme C terminus of SpoIIAB are both necessary and sufficient for this degradation . We also report that the use of the LCN extension as a degradation signal for ClpCP is unique to SpoIIAB .
Monitoring Gene Expression in Mixed Microbial Communities by Using DNA Microarrays. Philip Dennis, 2003.A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested . Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D . Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D . This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor . Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 105 cells/ml was clearly detected against a background of 108 cells/ml . Induction of two others (tfdB and tfdE) was detected from populations of 106 cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability . In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments . We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems . Role of the Bacillus methanolicus Citrate Synthase II Gene, citY, in Regulating the Secretion of Glutamate in L-Lysine-Secreting Mutants. Trygve Brautaset, 2003.The thermotolerant, restrictive methylotroph Bacillus methanolicus MGA3 (ATCC 53907) can secrete 55 g of glutamate per liter (maximum yield, 0.36 g/g) at 50°C with methanol as a carbon source and a source of ammonia in fed-batch bioreactors . A homoserine dehydrogenase mutant, 13A52-8A66, secreting up to 35 g of L-lysine per liter in fed-batch fermentations had minimal 2-oxoglutarate dehydrogenase activity [7.3 nmol min-1 (mg of protein)-1], threefold-increased pyruvate carboxylase activity [535 nmol min-1 (mg of protein)-1], and elevated citrate synthase (CS) activity [292 nmol min-1 (mg of protein)-1] and simultaneously secreted glutamate (20 to 30 g per liter) and L-lysine . The flow of carbon from oxaloacetate is split between transamination to aspartate and formation of citrate . To investigate the regulation of this branch point, the B . methanolicus gene citY encoding a CSII protein with activity at 50°C was cloned from 13A52-8A66 into a CS-deficient Escherichia coli K2-1-4 strain . A citY-deficient B . methanolicus mutant, NCS-L-7, was also isolated from the parent strain of 13A52-8A66 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, followed by selection with monofluoroacetate disks on glutamate plates . Characterization of these strains confirmed that citY in strain 13A52-8A66 was not altered and that B . methanolicus possessed several forms of CS . Analysis of citY cloned from NCS-L-7 showed that the reduced CS activity resulted from a frameshift mutation . The level of glutamate secreted by NCS-L-7 was reduced sevenfold and the ratio of L-lysine to glutamate secreted was increased 4.5-fold compared to the wild type in fed-batch cultures with glutamate feeding . This indicates that glutamate secretion in L-lysine-overproducing mutants can be altered in favor of increased L-lysine secretion by regulating in vivo CS activity .
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